Approximately 30,000 men a year die of prostate cancer (CaP) mostly due to the resistance of their tumors to androgen ablative therapy. The molecular process leading to the development of androgen independence (Al) is poorly understood. In this proposal we will investigate the molecular mechanisms underlying the development of AI. We hypothesize that multiple hAR-related alterations synergistically modify the functions of the androgen receptor (bAR) and lead to AT. To prove this, in these studies we will concentrate on three major types of hAR alterations: mutations of bAR, shortened CAG repeat lengths and co-activator over-expression. We will characterize the 55 known CaP-derived bAR mutations in a yeast functional assay model. At present only six have been submitted to such study. We will screen 100 metastatic CaP samples for the prevalence of bAR mutations using this yeast system. The known and newly identified hAR mutations will be analyzed for their transactivational activity, whether wild type (wt), loss-of-function (LOF), or gain-of-function (GOF) mutants. These mutants will also be evaluated for their ligand-binding specificity and DNA binding activity. We will next test the ability of shorter CAG repeat lengths and selected co-activators to modulate the function of wt and GOF mutant bARs. We will then transfect selected GOF mutant bARs into prostate epithelial cells to examine their biological functions (growth in soft agar and enhanced migration). At the end of this study, we expect to understand the influence of these bAR modifications on the functions of wt and mutant hARs, and will have shown that the yeast system can be used to rapidly and inexpensively screen CaPs for bAR mutations. We believe that defining the functions of these bAR-related alterations will aid in predicting prognosis and allow individualization of therapeutic regimes for patients who have CaP. Furthermore, the proposed studies are a logical step towards the development of new, improved treatments for advanced CaP.